This renewal proposal is designed to test the hypothesis that multiple host contacts and multiple blood feeding by mosquitoes in a single gonotrophic cycle are important factors in increasing the probability of arbovirus transmission. Although often documented, multiple feeding by mosquitoes generally has been overlooked. Our original proposal hypothesized that Cs. melanura could become infected with and transmit eastern equine encephalomyelitis (EEE) virus within one gonotrophic cycle. Results suggest that multiple feeding occurs during a brief time frame and that it increases the probability of an uninfected mosquito imbibing viremic blood and of virus transmission by an already infected mosquito. In this proposal we plan followup experiments to test intrinsic and extrinsic factors that might explain our findings of variation in multiple feeding. Preliminary studies with Ae. aegypti collected from a dengue (DEN) virus focus in Puerto Rico show that this mosquito commonly (24%) engages in multiple feeding. Studies are described that would determine more definitively how often and under what conditions this mosquito engages in multiple feeding and what effect multiple feeding has on DEN virus transmission. By using innovative histological, immunohistochemical, and blood marker techniques, we propose to determine more accurately the frequency of multiple feeding in the transmission of two important arboviruses (EEE and DEN). Results from our studies will provide insights into arboviral maintenance, help explain variation in time and space of arboviral disease outbreaks, and generate data useful in the design of disease prevention programs.